Ultrastructural changes in the bile canaliculi and the lateral surfaces of rat hepatocytes during regeneration following a two-third partial hepatectomy were studied by transmission and scanning electron microscopy. A marked increase in microvilli, widening of the intercellular spaces, and invagination and indentation of cytoplasmic membranes were seen in the lateral surfaces of hepatocytes during the early period after the operation. The density of the microvilli on the lateral surfaces gradually decreased and intercellular spaces returned to normal within 24 h, whereas the bile canaliculi revealed dilatation and tortuosity with elongation of microvilli. Hepatocytes during mitosis became rounded and showed dispersed microvilli on the sinusoidal surface. The bile canaliculi of mitotic hepatocytes were continuous with those of adjacent hepatocytes. On the 2nd or 3rd day posthepatectomy, hepatic plates became more than one-cell thick and hepatocytes showed occasional acinar arrangements around the dilated bile canalicular lumina. These features gradually returned to normal by one week after the operation. This study revealed unique sequential changes in the bile canaliculi and the lateral surfaces of hepatocytes during regeneration after partial hepatectomy.